Many problems in molecular-biological research and diagnostics require determining the amount or concentration of particular nucleic acids in a sample. For example, controlling the course of therapy in AIDS patients requires determining the number of copies of viral nucleic acids of the HIV virus (the “viral load”) in the blood. Since the nucleic acid to be determined is frequently present in a mixture with many other nucleic acids and, in addition, has a very low initial concentration, it is usually necessary to amplify and specifically detect the nucleic acid (or nucleic acids) to be determined.
There are various processes known in molecular biology for specific amplification (propagation) of nucleic acids, for example the polymerase chain reaction (PCR) which is described inter alia in U.S. Pat. No. 4,683,195. In addition, the PCR method is also suitable for quantifying nucleic acids, for example by including a known nucleic acid having a known initial concentration (of an internal standard) in the PCR reaction, wherefrom the initial concentration of other nucleic acids being detected in the sample can be inferred, as described in U.S. Pat. No. 5,219,727. Since in this case the concentration is determined only after the PCR reaction has been completed, this is referred to as an “end point method”.
In addition, methods have been developed which enable concentrations to be determined in the course of the PCR reaction and which are referred to as real time methods (real time PCR). Since, under ideal conditions, the number of the nucleic acids to be amplified can be doubled in each reaction cycle of the PCR, amplification is exponential. In real time PCR processes, the increase in concentration of the amplified nucleic acids can be monitored in real time, for example by incorporation of a fluorescent dye.
Further optical, mass-spectrometric and electrochemical processes for determining concentrations are also known.
Another process for amplifying nucleic acids is the ligase chain reaction (LCR) which is carried out similarly to a PCR but in which the enzyme used is a nucleic acid ligase, see, for example, Wiedmann et al. “Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction”, Appl Environ Microbiol. 1992, November; 58(11):3443-7.
Disadvantageously, however, these methods have a limited dynamic range. The typical dynamic range is between 1 to 100 and 1 to 1,000, but the range of nucleic acid concentrations to be measured often is distinctly larger, for example 1 to 1,000,000. The determination of concentrations therefore frequently requires serial dilutions to be made, for example 1 in 1, 1 in 10, 1 in 100, etc., in order to enable the concentration to be measured subsequently within the measurement range of the determination method. This is time-consuming and requires additional resources and is an additional source of error. A particular problem here is the sigmoidal time course profile of the concentration of the PCR product being produced, which is caused inter alia by the PCR reagents (primer, nucleoside triphosphates, etc.) being gradually used up with advanced reaction time (large number of PCR cycles), and/or the saturation zone of the detection method being reached. At this time, the concentration can no longer be determined in any meaningful sense.